Activation of HCV-specific T cells

ABSTRACT

The invention provides a method of activating hepatitis C virus (HCV)-specific T cells, including CD4 +  and CD8 +  T cells. HCV-specific T cells are activated using fusion proteins comprising HCV NS3, NS4, NS5a, and NS5b polypeptides, polynucleotides encoding such fusion proteins, or polypeptide or polynucleotide compositions containing the individual components of these fusions. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize a mammal against HCV.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to provisional patent applicationserial No. 60/161,713, filed Oct. 27, 1999, from which priority isclaimed under 35 USC §119(e)(1) and which is incorporated herein byreference in its entirety.

TECHNICAL AREA OF THE INVENTION

[0002] The invention relates to the activation of hepatitis Cvirus(HCV)-specific T cells. More particularly, the invention relates tothe use of multiple HCV polypeptides, either alone or as fusions, tostimulate cell-mediated immune responses, such as to activateHCV-specific T cells.

BACKGROUND OF THE INVENTION

[0003] Hepatitis C virus (HCV) infection is an important health problemwith approximately 1% of the world's population infected with the virus.Over 75% of acutely infected individuals eventually progress to achronic carrier state that can result in cirrhosis, liver failure, andhepatocellular carcinoma. See Alter et al. (1992) N. Engl. J. Med.327:1899-1905; Resnick and Koff. (1993) Arch. Intern. Med.153:1672-1677; Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al.(1995) N. Engl. J. Med. 332:1463-1466.

[0004] Despite extensive advances in the development of pharmaceuticalsagainst certain viruses like HIV, control of acute and chronic HCVinfection has had limited success (Hoofnagle and di Bisceglie (1997) N.Engl. J. Med. 336:347-356). In particular, generation of a strongcytotoxic T lymphocyte (CTL) response is thought to be important for thecontrol and eradication of HCV infections. Thus, there is a need in theart for effective methods of inducing strong CTL responses against HCV.

SUMMARY OF THE INVENTION

[0005] It is an object of the invention to provide reagents and methodsfor activating T cells which recognize epitopes of HCV polypeptides.This and other objects of the invention are provided by one or more ofthe embodiments described below.

[0006] The invention provides HCV proteins useful for activatingHCV-specific T cells. One embodiment provides a fusion protein thatconsists essentially of an NS3, an NS4, and an NS5a polypeptide.

[0007] Another embodiment provides a fusion protein that consistsessentially of an NS3, an NS4, an NS5a, and NS5b polypeptide of an HCV.

[0008] Still another embodiment of the invention provides a fusionprotein comprising an NS3, an NS4, an NS5a and optionally an NS5bpolypeptide of an HCV. One of the HCV polypeptides is derived from adifferent strain of HCV than the other polypeptides.

[0009] The invention also provides compositions comprising any of thesefusion proteins and a pharmaceutically acceptable carrier.

[0010] Another embodiment provides a composition consisting essentiallyof an NS3, an NS4, and an NS5a polypeptide, or a composition consistingessentially of polynucleotides encoding the individual proteins.

[0011] Another embodiment provides a composition that consistsessentially of an NS3, an NS4, an NS5a, and NS5b polypeptide of an HCV,or a composition consisting essentially of polynucleotides encoding theindividual proteins.

[0012] Still another embodiment of the invention provides a compositionconsisting essentially of an NS3, an NS4, an NS5a and optionally an NS5bpolypeptide of an HCV, or a composition consisting essentially ofpolynucleotides encoding the individual proteins. One of the HCVpolypeptides or polynucleotides is derived from a different strain ofHCV than the others.

[0013] Even another embodiment of the invention provides an isolated andpurified polynucleotide which encodes a fusion protein consistingessentially of an NS3, an NS4, and an NS5a polypeptide of an HCV or afusion protein consisting essentially of an NS3, an NS4, an NS5a, and anNS5b polypeptide of an HCV.

[0014] Yet another embodiment of the invention provides a compositioncomprising an isolated and purified polynucleotide which encodes afusion protein consisting essentially of either an NS3, an NS4, and anNS5a polypeptide of an HCV or consisting essentially of an NS3, an NS4,an NS5a, and an NS5b polypeptide of an HCV. The composition alsocomprises a pharmaceutically acceptable carrier.

[0015] Another embodiment of the invention provides isolated andpurified polynucleotide which encodes a fusion protein comprising anNS3, an NS4, and an NS5a polypeptide of an HCV in which one of the NS3,NS4, and NS5a polypeptides is derived from a different strain of HCVthan the other two polypeptides. The invention also provides acomposition comprising this polynucleotide and a pharmaceuticallyacceptable carrier.

[0016] Yet another embodiment of the invention provides an isolated andpurified polynucleotide which encodes a fusion protein comprising anNS3, an NS4, an NS5a, and an NS5b polypeptide of an HCV. One of thepolypeptides is derived from a different strain of HCV than the otherpolypeptides. The invention also provides a composition comprising thispolynucleotide and a pharmaceutically acceptable carrier.

[0017] Even another embodiment of the invention provides a method ofactivating T cells which recognize an epitope of an HCV polypeptide. Tcells are contacted with a fusion protein comprising an NS3, an NS4, andan NS5a polypeptide of an HCV. A population of activated T cellsrecognizes an epitope of the NS3, NS4, or NS5a polypeptide.Alternatively, T cells are contacted with a fusion protein comprising anHCV NS3, NS4, NS5a, NS5b-polypeptide of an HCV. A population ofactivated T.cells recognizes an epitope of the NS3, NS4, NS5a, or NS5bpolypeptide.

[0018] The invention thus provides methods and reagents for activating Tcells which recognize epitopes of HCV polypeptides. These methods andreagents are particularly advantageous for identifying epitopes of HCVpolypeptides associated with a strong CTL response and for immunizingmammals, including humans, against HCV.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 shows a side-by-side comparison of IFN-γ expressiongenerated in animals in response to delivery of alphavirus constructsencoding NS3NS4NS5a.

[0020]FIG. 2 shows IFN-γ expression generated in animals in response todelivery of plasmid DNA encoding NS3NS4NS5a (“naked”), PLG-linked DNAencoding NS3NS4NS5a (“PLG), separate DNA plasmids encoding NS5a, NS34a,and NS4ab (“naked”), and PLG-linked DNA encoding NS5a, NS34a, and NS4ab(“PLG”).

DETAILED DESCRIPTION OF THE INVENTION

[0021] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of chemistry, biochemistry,recombinant DNA techniques and immunology, within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition);Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press,.Inc.); DNA Cloning, Vols. I and II (D. N. Glover ed.); OligonucleotideSynthesis (M. J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins eds.); Animal Cell Culture (R. K. Freshney ed.); Perbal, B.,A Practical Guide to Molecular Cloning.

[0022] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0023] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “an antigen” includes a mixture of two or moreantigens, and the like.

[0024] I. Definitions

[0025] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0026] The terms “polypeptide” and “protein” refer to a polymer of aminoacid residues and are not limited to a minimum length of the product.Thus, peptides, oligopeptides, dimers, multimers, and the like, areincluded within the definition. Both full-length proteins and fragmentsthereof are encompassed by the definition. The terms also includepostexpression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence, so long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

[0027] An HCV polypeptide is a polypeptide, as defined above, derivedfrom the HCV polyprotein. The polypeptide need not be physically derivedfrom HCV, but may be synthetically or recombinantly produced. Moreover,the polypeptide may be derived from any of the various HCV strains, suchas from strains 1, 2, 3 or 4 of HCV. A number of conserved and variableregions are known between these strains and, in general, the amino acidsequences of epitopes derived from these regions will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, preferably more than 40%, when the two sequences are aligned. Thus,for example, the term “NS4” polypeptide refers to native NS4 from any ofthe various HCV strains, as well as NS4 analogs, muteins and immunogenicfragments, as defined further below.

[0028] The terms “analog” and “mutein” refer to biologically activederivatives of the reference molecule, or fragments of such derivatives,that retain desired activity, such as the ability to stimulate acell-mediated immune response, as defined below. In general, the term“analog” refers to compounds having a native polypeptide sequence andstructure with one or more amino acid additions, substitutions(generally conservative in nature) and/or deletions, relative to thenative molecule, so long as the modifications do not destroy immunogenicactivity. The term “mutein” refers to peptides having one or morepeptide mimics (“peptoids”), such as those described in InternationalPublication No. WO 91/04282. Preferably, the analog or mutein has atleast the same immunoactivity as the native molecule. Methods for makingpolypeptide analogs and muteins are known in the art and are describedfurther below.

[0029] Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

[0030] By “fragment” is intended a polypeptide consisting of only a partof the intact full-length polypeptide sequence and structure. Thefragment can include a C-terminal deletion and/or an N-terminal deletionof the native polypeptide. An “immunogenic fragment” of a particular HCVprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains immunogenic activity, as measured by theassays described herein. For a description of various HCV epitopes, see,e.g., Chien et al., Proc. Natl Acad. Sci. USA (1992) 89:10011-10015;Chien et al., J Gastroent. Hepatol. (1993) 8:S33-39; Chien et al.,International Publication No. WO 93/00365; Chien, D. Y., InternationalPublication No. WO 94/01778; commonly owned, allowed U.S. patentapplication Ser. Nos. 08/403,590 and 08/444,818.

[0031] The term “epitope” as used herein refers to a sequence of atleast about 3 to 5, preferably about 5 to 10 or 15, and not more thanabout 1,000 amino acids (or any integer therebetween), which define asequence that by itself or as part of a larger sequence, binds to anantibody generated in response to such sequence. There is no criticalupper limit to the length of the fragment, which may comprise nearly thefull-length of the protein sequence, or even a fusion protein comprisingtwo or more epitopes from the HCV polyprotein. An epitope for use in thesubject invention is not limited to a polypeptide having the exactsequence of the portion of the parent protein from which it is derived.Indeed, viral genomes are in a state of constant flux and containseveral variable domains which exhibit relatively high degrees ofvariability between isolates. Thus the term “epitope” encompassessequences identical to the native sequence, as well as modifications tothe native sequence, such as deletions, additions and substitutions(generally conservative in nature).

[0032] Regions of a given polypeptide that include an epitope can beidentified using any number of epitope mapping techniques, well known inthe art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al.(1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

[0033] As used herein, the term “conformational epitope” refers to aportion of a full-length protein, or an analog or mutein thereof, havingstructural features native to the amino acid sequence encoding theepitope within the full-length natural protein. Native structuralfeatures include, but are not limited to, glycosylation and threedimensional structure. Preferably, a conformational epitope is producedrecombinantly and is expressed in a cell from which it is extractableunder conditions which preserve its desired structural features, e.g.without denaturation of the epitope. Such cells include bacteria, yeast,insect, and mammalian cells. Expression and isolation of recombinantconformational epitopes from the HCV polyprotein are described in e.g.,International Publication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO92/08734, which applications are herein incorporated by reference intheir entirety.

[0034] An “immunological response” to an HCV antigen (including bothpolypeptide and polynucleotides encoding polypeptides that are expressedin vivo) or composition is the development in a subject of a humoraland/or a cellular immune response to molecules present in thecomposition of interest. For purposes of the present invention, a“humoral immune response” refers to an immune response mediated byantibody molecules, while a “cellular immune response” is one mediatedby T-lymphocytes and/or other white blood cells. One important aspect ofcellular immunity involves an antigen-specific response by cytolyticT-cells (“CTLs”). CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes, Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

[0035] A composition or vaccine that elicits a cellular immune responsemay serve to sensitize a vertebrate subject by the presentation ofantigen in association with MHC molecules at the cell surface. Thecell-mediated immune response is directed at, or near, cells presentingantigen at their surface. In addition, antigen-specific T-lymphocytescan be generated to allow for the future protection of an immunizedhost.

[0036] The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

[0037] Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T- cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection oralleviation of symptoms to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

[0038] A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A transcription termination sequence may belocated 3′ to the coding sequence.

[0039] A “nucleic acid” molecule or “polynucleotide” can include bothdouble- and single-stranded sequences and refers to, but is not limitedto, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNAsequences from viral (e.g. DNA viruses and retroviruses) or procaryoticDNA, and especially synthetic DNA sequences. The term also capturessequences that include any of the known base analogs of DNA and RNA.

[0040] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform theirdesired function. Thus, a given promoter operably linked to a codingsequence is capable of effecting the expression of the coding sequencewhen the proper transcription factors, etc., are present. The promoterneed not be contiguous with the coding sequence, so long as it functionsto direct the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

[0041] “Recombinant” as used herein to describe a nucleic acid moleculemeans a -polynucleotide-of genomic, cDNA, viral, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation is notassociated with all or a portion of the polynucleotide with which it isassociated in nature. The term “recombinant” as used with respect to aprotein or polypeptide means a polypeptide produced by expression of arecombinant polynucleotide. In general, the gene of interest is clonedand then expressed in transformed organisms, as described further below.The host organism expresses the foreign gene to produce the proteinunder expression conditions.

[0042] A “control element” refers to a polynucleotide sequence whichaids in the expression of a coding sequence to which it is linked. Theterm includes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

[0043] A “promoter” as used herein is a DNA regulatory region capable ofbinding RNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

[0044] A control sequence “directs the transcription” of a codingsequence in a cell when RNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA, which is then translatedinto the polypeptide encoded by the coding sequence.

[0045] “Expression cassette” or “expression construct” refers to anassembly which is capable of directing the expression of the sequence(s)or gene(s) of interest. The expression cassette includes controlelements, as described above, such as a promoter which is operablylinked to (so as to direct transcription of) the sequence(s) or gene(s)of interest, and often includes a polyadenylation sequence as well.Within certain embodiments of the invention, the expression cassettedescribed herein may be contained within a plasmid construct. Inaddition to the components of the expression cassette, the plasmidconstruct may also include, one or more selectable markers, a signalwhich allows the plasmid construct to exist as single-stranded DNA(e.g., a M13 origin of replication), at least one multiple cloning site,and a “mammalian” origin of replication (e.g., a SV40 or adenovirusorigin of replication).

[0046] “Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

[0047] A “host cell” is a cell which has been transformed, or is capableof transformation, by an exogenous DNA sequence.

[0048] By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macromolecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

[0049] The term “purified” as used herein preferably means at least 75%by weight, more preferably at least 85% by weight, more preferably stillat least 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type are present.

[0050] “Homology” refers to the percent identity between twopolynucleotide or two polypeptide moieties. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 50% , preferably at least-about 75%,more preferably at least about 80%-85%, preferably at least about 90%,and most preferably at least about 95%-98%, or more, sequence identityover a defined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified DNA or polypeptide sequence.

[0051] In general, “identity”. refers to an exactnucleotide-to-nucleotide or amino acid-to-amino acid correspondence oftwo polynucleotides or polypeptide sequences, respectively. Percentidentity can be determined by a direct comparison of the sequenceinformation between two molecules by aligning the sequences, countingthe exact number of matches, between the two aligned sequences, dividingby the length of the shorter sequence, and multiplying the result by100. Readily available computer programs can be used to aid in theanalysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence andStructure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedicalResearch Foundation, Washington, D.C., which adapts the local homologyalgorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981for peptide analysis. Programs for determining nucleotide sequenceidentity are available in the Wisconsin Sequence Analysis Package,Version 8 (available from Genetics Computer Group, Madison, Wis.) forexample, the BESTFIT, FASTA and GAP programs, which also rely on theSmith and Waterman algorithm. These programs are readily utilized withthe default parameters recommended by the manufacturer and described inthe Wisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

[0052] Another method of establishing percent identity in the context ofthe present invention is to use the MPSRCH package of programscopyrighted by the University of Edinburgh, developed by John F. Collinsand Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (MountainView, Calif.). From this suite of packages the Smith-Waterman algorithmcan be employed where default parameters are used for the scoring table(for example, gap open penalty of 12, gap extension penalty of one, anda gap of six). From the data generated the “Match” value reflects“sequence identity.” Other suitable programs for calculating the percentidentity or similarity between sequences are generally known in the art,for example, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter none; strandboth; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences;sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0053] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

[0054] By “nucleic acid immunization” is meant the introduction of anucleic acid molecule encoding one or more selected antigens into a hostcell, for the in vivo expression of the antigen or antigens. The nucleicacid molecule can be introduced directly into the recipient subject,such as by injection, inhalation, oral, intranasal and mucosaladministration, or the like, or can be introduced ex vivo, into cellswhich have been removed from the host. In the latter case, thetransformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

[0055] As used herein, “treatment” refers to any of (i) the preventionof infection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

[0056] By “vertebrate subject” is meant any member of the subphylumcordata, including, without limitation, humans and other primates,including non-human primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs; birds, includingdomestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like. The term: does notdenote a particular age. Thus, both adult and newborn individuals areintended to be covered. The invention described herein is intended foruse in any of the above vertebrate species, since the immune systems ofall of these vertebrates operate similarly.

[0057] II. Modes of Carrying out the Invention

[0058] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

[0059] Although a number of compositions and methods similar orequivalent to those described herein can be used in the practice of thepresent invention, the preferred materials and methods are describedherein.

[0060] It is a discovery of the present invention that fusion proteins,combinations of the individual components of these fusions, andpolynucleotides encoding the same, comprising an NS3, an NS4, and anNS5a polypeptide or an NS3, an NS4, an NS5a, and an NS5b polypeptide ofan HCV virus can be used to activate HCV-specific T cells, i.e., T cellswhich recognize epitopes of these polypeptides. Activation ofHCV-specific T cells by such fusion polypeptides and proteins, orcombinations of the individual polypeptides that make up the fusions,provide both in vitro and in vivo model systems for the development ofHCV vaccines, particularly for identifying HCV polypeptide epitopesassociated with a response. The fusion proteins, or combinations of theindividual proteins, can also be used to generate an immune responseagainst HCV in a mammal, particularly a CTL response for eithertherapeutic or prophylactic purposes.

[0061] NS3NS4NS5a and NS3NS4NS5aNS5b Fusion Proteins

[0062] The genomes of HCV strains contain a single open reading frame ofapproximately 9,000 to 12,000 nucleotides, which is transcribed into apolyprotein. An HCV polyprotein is cleaved to produce at least tendistinct products, in the order ofNH₂-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Fusion proteins ofthe invention (NS3NS4NS5a fusion proteins, also termed “NS345a” herein)comprise HCV NS3, NS4 (NS4a and NS4b), and NS5a polypeptides or compriseHCV NS3, NS4 (NS4a and NS4b), NS5a, and NS5b polypeptides(NS3NS4NS5aNS5b fusion proteins, also termed “NS345ab” herein). Otherfusions described in the examples include fusions of HCV NS3 and NS4(NS4a and NS4b, also termed “NS34” and “NS34ab”), as well as fusions ofHCV NS3 and NS4a (also termed “NS34a”).

[0063] The HCV NS3 protein functions as a protease and a helicase andoccurs at approximately amino acid 1027 to amino acid 1657 of thepolyprotein (numbered relative to HCV-1). See Choo et al. (1991) Proc.Natl. Acad. Sci. USA 88:2451-2455. HCV NS4 occurs at approximately aminoacid 1658 to amino acid 1972, NS5a occurs at approximately amino acid1973 to amino acid 2420, and HCV NS5b occurs at approximately amino acid2421 to amino acid 3011 of the polyprotein (numbered relative to HCV-1)(Choo et al., 1991).

[0064] The NS3, NS4, NS5a, and NS5b polypeptides present in the variousfusions described above can either be full-length polypeptides orportions of NS3, NS4 (NS4a and NS4b), NS5a, and NS5b polypeptides. Theportions of NS3, NS4, NS5a, and NS5b polypeptides making up the fusionprotein comprise at least one epitope, which is recognized by a T cellreceptor on an activated T cell, such as 2152-HEYPVGSQL-2160 (SEQ IDNO:1) and 2224-AELIEANLLWRQEMG-2238 (SEQ ID NO:2). Epitopes of NS3, NS4(NS4a and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can beidentified-by several methods. For example, NS3, NS4, NS5a, NS5bpolypeptides or fusion proteins comprising any combination of the above,can be isolated, for example, by immunoaffinity purification using amonoclonal antibody for the polypeptide or protein. The isolated proteinsequence can then be screened by preparing a series of short peptides byproteolytic cleavage of the purified protein, which together span theentire protein sequence. By starting with, for example, 100-merpolypeptides, each polypeptide can be tested for the presence ofepitopes recognized by a T cell receptor on an HCV-activated T cell,progressively smaller and overlapping fragments can then be tested froman identified 100-mer to map the epitope of interest.

[0065] Epitopes recognized by a T cell receptor on an HCV-activated Tcell can be identified by, for example, ⁵¹Cr release assay (see Example2) or by lymphoproliferation assay (see Example 4). In a ⁵¹Cr releaseassay, target cells can be constructed that display the epitope ofinterest by cloning a polynucleotide encoding the epitope into anexpression vector and transforming the expression vector into the targetcells. HCV-specific CD8⁺ T cells will lyse target cells displaying anNS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitope and will notlyse cells that do not display such an epitope. In anlymphoproliferation assay, HCV-activated CD4⁺ T cells will proliferatewhen cultured with an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, orNS3NS4NS5aNS5b epitopic peptide, but not in the absence of an HCVepitopic peptide.

[0066] NS3, NS4, NS5a, and NS5b polypeptides can occur in any order inthe fusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10or more of one or more of the polypeptides may occur in the fusionprotein. Multiple viral strains of HCV occur, and NS3, NS4, NS5a, andNS5b polypeptides of any of these strains can be used in a fusionprotein.

[0067] Nucleic acid and amino acid sequences of a number of HCV strainsand isolates, including nucleic acid and amino acid sequences of NS3,NS4, NS5a, NS5b genes and polypeptides have been determined. Forexample, isolate HCV J1.1 is described in Kubo et al. (1989) Japan.Nucl. Acids Res. 17:10367-10372; Takeuchi et al.(1990) Gene 91:287-291;Takeuchi et al. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al.(1990) Nucl. Acids Res. 18:4626. The complete coding sequences of twoindependent isolates, HCV-J and BK, are described by Kato et al., (1990)Proc. Natl. Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J.Virol. 65:1105-1113 respectively.

[0068] Publications that describe HCV-1 isolates include Choo et al.(1990) Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad.Sci. USA 88:2451-2455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA88:1711-1715. HCV isolates HC-J1 and HC-J4 are described in Okamotoet.al (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT 18˜, HCT23, Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991)Virol. 180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 are describedin Enomoto et al. (1990) Biochem. Biophys. Res. Commun. 170:1021-1025.HCV isolates A, C, D & E are described in Tsukiyama-Kohara et al. (1991)Virus Genes 5:243-254.

[0069] Each of the NS3, NS4, NS5a, and NS5b components of a fusionprotein can be obtained from the same HCV strain or isolate or fromdifferent HCV strains or isolates. Fusion proteins comprising HCVpolypeptides from, for example, the NS3 polypeptide can be derived froma first strain of HCV, and the NS4, and NS5a polypeptides can be derivedfrom a second strain of HCV. Alternatively, the NS4 polypeptide can bederived from a first strain of HCV, and the NS3 and NS5a polypeptidescan be derived from a second strain of HCV. Optionally, the NS5apolypeptide can be derived from a first strain of HCV, and the NS3 andNS4 polypeptides can be derived from a second strain of HCV. NS3, NS4and NS5a polypeptides that are each be derived from different HCVstrains can also be used in an NS3NS4NS5a fusion protein. Similarly, ina fusion protein comprising NS5b, at least one of the NS3, NS4, NS5a,and NS5b polypeptides can be derived from a different HCV strain thanthe other polypeptides. Optionally, NS3, NS4, NS5a, and NS5bpolypeptides that are each derived from different HCV strains can alsobe used in an NS3NS4NS5aNS5b fusion protein.

[0070] In addition to NS3, NS4a, NS4b, NS5a and NS5b, the fusionproteins can contain other polypeptides derived from the HCVpolyprotein. For example, it may be desirable to include polypeptidesderived from the core region of the HCV polyprotein. This region occursat amino acid positions 1-191 of the HCV polyprotein, numbered relativeto HCV-1. Either the full-length protein or epitopes of the full-lengthprotein may be used in the subject fusions, such as those epitopes foundbetween amino acids 10-53, amino acids 10-45, amino acids 67-88, aminoacids 120-130, or any of the core epitopes identified in, e.g., Houghtonet al., U.S. Pat. No. 5,350,671; Chien et al., Proc. Natl. Acad. Sci.USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993)8:S33-39; Chien et al., International Publication No. WO 93/00365;Chien, D. Y., International Publication No. WO 94/01778; and commonlyowned, allowed U.S. patent application Ser. Nos. 08/403,590 and08/444,818, the disclosures of which are incorporated herein byreference in their entireties.

[0071] Preferably, the above-described fusion proteins, as well as theindividual components of these proteins, are produced recombinantly. Apolynucleotide encoding these proteins can be introduced into anexpression vector which can be expressed in a suitable expressionsystem. A variety of bacterial, yeast, mammalian and insect expressionsystems are available in the art and any such expression system can beused. Optionally, a polynucleotide encoding these proteins can betranslated in a cell-free translation system. Such methods are wellknown in the art. The proteins also can be constructed by solid phaseprotein synthesis.

[0072] If desired, the fusion proteins, or the individual components ofthese proteins, also can contain other amino acid sequences, such asamino acid linkers or signal sequences, as well as ligands useful inprotein purification, such as glutathione-S-transferase andstaphylococcal protein A.

[0073] NS3NS4NS5a and NS3NS4NS5aNS5b Polynucleotides

[0074] Polynucleotides contain less than an entire HCV genome and can beRNA or single- or double-stranded DNA. Preferably, the polynucleotidesare isolated free of other components, such as proteins and lipids.NS3NS4NS5a polynucleotides encode the NS3NS4NS5a fusion proteinsdescribed above, and thus comprise coding sequences for NS3, NS4, andNS5a polypeptides. NS3NS4NS5aNS5b polynucleotides encode theNS3NS4NS5aNS5b fusion proteins described above, and thus comprise codingsequences for NS3, NS4, NS5a, and NS5b polypeptides. Similarly,polynucleotides encoding other fusions, such as NS3NS4 and NS3NS4a willcomprise sequences for the individual HCV polypeptides. Polynucleotidesof the invention can also comprise other nucleotide sequences, such assequences coding for linkers, signal sequences, or ligands useful inprotein purification such as glutathione-S-transferase andstaphylococcal protein A.

[0075] Polynucleotides encoding NS3, NS4, NS5a and/or NS5b can beisolated from a genomic library derived from nucleic. acid sequencespresent in, for example, the plasma, serum, or liver homogenate of anHCV infected individual or can be synthesized in the laboratory, forexample, using an automatic synthesizer. An amplification method such asPCR can be used to amplify polynucleotides from either HCV genomic DNAor cDNA encoding NS3, NS4, NS5a, or NS5b.

[0076] Polynucleotides encoding NS3, NS4, NS5a, or NS5b polypeptides cancomprise coding sequences for these polypeptides which occur naturallyor can be artificial sequences which do not occur in nature. Thesepolynucleotides can be ligated to form a coding sequence for the fusionproteins using standard molecular biology techniques. If desired,polynucleotides can be cloned into an expression vector and transformedinto, for example, bacterial, yeast, insect, or mammalian cells so thatthe fusion proteins of the invention can be expressed in and isolatedfrom a cell culture.

[0077] The expression constructs of the present invention, including thedesired fusion, or individual expression constructs comprising theindividual components of these fusions, may be used for nucleic acidimmunization, to activate HCV-specific T cells, using standard genedelivery protocols. Methods for gene delivery are known in the art. See,e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. Genes can be delivered eitherdirectly to the vertebrate subject or, alternatively, delivered ex vivo,to cells derived from the subject and the cells reimplanted in thesubject. For example, the constructs can be delivered as plasmid DNA,e.g., contained within a plasmid, such as pBR322, pUC, or ColE1

[0078] Additionally, the expression constructs can be packaged inliposomes prior to delivery to the cells. Lipid encapsulation isgenerally accomplished using liposomes which are able to stably bind orentrap and retain nucleic acid. The ratio of condensed DNA to lipidpreparation can vary but will generally be around 1:1 (mg DNA:micromoleslipid), or more of lipid. For a review of the use of liposomes ascarriers for delivery of nucleic acids, see, Hug and Sleight, Biochim.Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods ofEnzymology (1983), Vol. 101, pp. 512-527.

[0079] Liposomal preparations for use with the present invention includecationic (positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can beprepared from readily available materials using techniques well known inthe art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978)75:4194-4198; PCT Publication No. WO 90/11092 for a description of thesynthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. The various liposome-nucleic acid complexes are preparedusing methods known in the art. See, e.g., Straubinger et al., inMETHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc.Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al.,Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979)17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostroet al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al.,Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittnatter,Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem.(1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA(1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0080] The DNA can also be delivered in cochleate lipid compositionssimilar to those described by Papahadjopoulos et al., Biochem. Biophys.Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and4,871,488.

[0081] A number of viral based systems have been developed for genetransfer into mammalian cells. For example, retroviruses provide aconvenient platform for gene delivery systems, such as murine sarcomavirus, mouse mammary tumor virus, Moloney murine leukemia virus, andRous sarcoma virus. A selected gene can be inserted into a vector andpackaged in retroviral particles using techniques known in the art. Therecombinant virus can,then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of retroviral systems havebeen described (U.S. Pat. No. 5,219,740; Miller and Rosman,BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990)1:5-14; Scarpa et al., Virology (1991) 180:849-852; Bums et al., Proc.Natl. Acad Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin,Cur. Opin. Genet. Develop. (1993) 3:102-109. Briefly, retroviral genedelivery vehicles of the present invention may be readily constructedfrom a wide variety of retroviruses, including for example, B, C, and Dtype retroviruses as well as spumaviruses and lentiviruses such as FIV,HIV, HIV-1, HIV-2 and SIV (see RNA Tumor Viruses, Second Edition, ColdSpring Harbor Laboratory, 1985). Such retroviruses may be readilyobtained from depositories or collections such as the American TypeCulture Collection (“ATCC”; 10801 University Blvd., Manassas, Va.20110-2209), or isolated from known sources using commonly availabletechniques.

[0082] A number of adenovirus vectors have also been described, such asadenovirus Type 2 and Type 5 vectors. Unlike retroviruses whichintegrate into the host genome, adenoviruses persist extrachromosomallythus minimizing the risks associated with insertional mutagenesis(Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J.Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994)5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., GeneTherapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629;and Rich et al., Human Gene Therapy (1993) 4:461-476).

[0083] Molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery.

[0084] Members of the Alphavirus genus, such as but not limited tovectors derived from the Sindbis and Semliki Forest viruses, VEE, willalso find use as viral vectors for delivering the gene of interest. Fora description of Sindbis-virus derived vectors useful for the practiceof the instant methods, see, Dubensky et al., J. Virol. (1996)70.508-519; and International Publication Nos. WO 95/07995 and WO96/17072.

[0085] Other vectors can be used, including but not limited to simianvirus 40, cytomegalovirus. Bacterial vectors, such as Salmonella ssp.Yersinia enterocolitica, Shigella spp., Vibrio cholerae, Mycobacteriumstrain BCG, and Listeria monocytogenes can be used. Minichromosomes suchas MC and MCl, bacteriophages, cosmids plasmids into which phage lambdacos sites have been inserted) and replicons (genetic elements that arecapable of replication under their own control in a cell) can also beused.

[0086] The expression constructs may also be encapsulated, adsorbed to,or associated with, particulate carriers. Such carriers present multiplecopies of a selected molecule to the immune system and promote trappingand retention of molecules in local lymph nodes. The particles can bephagocytosed by macrophages and can enhance antigen presentation throughcytokine release. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al.,J. Microencap. (1996).

[0087] A wide variety of other methods can be used to deliver theexpression constructs to cells. Such methods include DEAEdextran-mediated transfection, calcium phosphate precipitation,polylysine- or polyornithine-mediated transfection, or precipitationusing other insoluble inorganic salts, such as strontium phosphate,aluminum silicates including bentonite and kaolin, chromic oxide,magnesium silicate, talc, and the like. Other useful methods oftransfection include electroporation, sonoporation, protoplast fusion,liposomes, peptoid delivery, or microinjection. See, e.g., Sambrook etal., supra, for a discussion of techniques for transforming cells ofinterest; and Felgner, P. L., Advanced Drug Delivery Reviews(1990)5:163-187, for a review of delivery systems useful for genetransfer. One particularly effective method of delivering DNA usingelectroporation is described in International Publication No.WO/0045823.

[0088] Additionally, biolistic delivery systems employing particulatecarriers such as gold and tungsten, are especially useful for deliveringthe expression constructs of the present invention. The particles arecoated with the construct to be delivered and accelerated to highvelocity, generally under a reduced atmosphere, using a gun powderdischarge from a “gene gun.” For a description of such techniques, andapparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050;5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744.

[0089] Compositions Comprising Fusion Proteins or Polynucleotides

[0090] The invention also provides compositions comprising the fusionproteins or polynucleotides, as well as compositions including theindividual components of these fusion proteins or polynucleotides.Compositions of the invention preferably comprise a pharmaceuticallyacceptable carrier. The carrier should not itself induce the productionof antibodies harmful to the host. Pharmaceutically acceptable carriersare well known to those in the art. Such carriers include, but are notlimited to, large, slowly metabolized, macromolecules, such as proteins,polysaccharides such as latex functionalized sepharose, agarose,cellulose, cellulose beads and the like, polylactic acids, polyglycolicacids, polymeric amino acids such as polyglutamic acid, polylysine, andthe like, amino acid copolymers, and inactive virus particles.

[0091] Pharmaceutically acceptable salts can also be used incompositions of the invention, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates, or sulfates, as well as saltsof organic acids such as acetates, proprionates, malonates, orbenzoates. Especially useful protein substrates are serum albumins,keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,ovalbumin, tetanus toxoid, and other proteins well known to those ofskill in the art. Compositions of the invention can also contain liquidsor excipients, such as water, saline, glycerol, dextrose, ethanol, orthe like, singly or in combination, as well as substances such aswetting agents, emulsifying agents, or pH buffering agents. Liposomescan also be used as a carrier for a composition of the invention, suchliposomes are described above.

[0092] If desired, co-stimulatory molecules which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asGM-CSF, IL-2, and. IL- 12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants which can be used include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80,and 0.5% Span 85 (optionally containing various amounts of MTP-PE ),formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5)cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; (6)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, e.g.,WO 93/13302 and WO 92/19265; (7) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition; and (8) microparticles with adsorbed macromolecules, asdescribed in copending U.S. patent application Ser. No. 09/285,855(filed Apr. 2, 1999) and international patent application Ser. No.PCT/US99/17308 (filed Jul. 29, 1999). Alum and MF59 are preferred.

[0093] As mentioned above, muramyl peptides include, but are not limitedto, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred tonor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), etc.

[0094] Methods of Producing HCV-Specific Antibodies

[0095] The HCV fusion proteins, such as NS3NS4SN5a and NS3NS4NS5aNS5bfusion proteins, can be used to produce HCV-specific polyclonal andmonoclonal antibodies. HCV-specific polyclonal and monoclonal antibodiesspecifically bind to HCV antigens.

[0096] Polyclonal antibodies can be produced by administering the fusionprotein to a mammal, such as a mouse, a rabbit, a goat, or a horse.Serum from the immunized animal is collected and the antibodies arepurified from the plasma by, for example, precipitation with ammoniumsulfate, followed by chromatography, preferably affinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art.

[0097] Monoclonal antibodies directed against HCV-specific epitopespresent in the fusion proteins can also be readily produced. Normal Bcells from a mammal, such as a mouse, immunized with, e.g., anNS3NS4SN5a or an NS3NS4NS5aNS5b fusion protein can be fused with, forexample, HAT-sensitive mouse myeloma cells to produce hybridomas.Hybridomas producing HCV-specific antibodies can be identified using RIAor ELISA and isolated by cloning in semi-solid agar or by limitingdilution. Clones producing HCV-specific antibodies are isolated byanother round of screening.

[0098] Antibodies, either monoclonal and polyclonal, which are directedagainst HCV epitopes, are particularly useful for detecting the presenceof HCV or HCV antigens in a sample, such as a serum sample from anHCV-infected human. An immunoassay for an HCV antigen may utilize oneantibody or several antibodies. An immunoassay for an HCV antigen mayuse, for example, a monoclonal antibody directed towards an HCV epitope,a combination of monoclonal antibodies directed towards epitopes of oneHCV polypeptide, monoclonal antibodies directed towards epitopes ofdifferent HCV polypeptides, polyclonal antibodies directed towards thesame HCV antigen, polyclonal antibodies directed towards different HCVantigens, or a combination of monoclonal and polyclonal antibodies.Immunoassay protocols may be based, for example, upon competition,direct reaction, or sandwich type assays using, for example, labeledantibody. The labels may be, for example, fluorescent, chemiluminescent,or radioactive.

[0099] The polyclonal or monoclonal antibodies may further be used toisolate HCV particles or antigens by immunoaffinity columns. Theantibodies can be affixed to a solid support by, for example, adsorptionor by covalent linkage so that the antibodies retain theirimmunoselective activity. Optionally, spacer groups may be included sothat the antigen binding site of the antibody remains accessible. Theimmobilized antibodies can then be used to bind HCV particles orantigens from a biological sample, such as blood or plasma. The boundHCV particles or antigens are recovered from the column matrix by, forexample, a change in pH.

[0100] HCV-Specific T Cells

[0101] HCV-specific T cells that are activated by the above-describedfusions, including the NS3NS4NS5a fusion protein or NS3NS4NS5aNS5bfusion protein, expressed in vivo or in vitro, or combinations of theindividual components of the fusions, preferably recognize an epitope ofan HCV polypeptide such as an NS3, NS4, NS5a, NS5b polypeptide,including an epitope of an NS3NS4NS5a fusion protein or anNS3NS4NS5aNS5b fusion protein. HCV-specific T cells can be CD8⁺ or CD4⁺.

[0102] HCV-specific CD8⁺ T cells preferably are cytotoxic T lymphocytes(CTL) which can kill HCV-infected cells that display NS3, NS4, NS5a,NS5b epitopes complexed with an MHC class I molecule. HCV-specific CD8⁺T cells may also express interferon-γ (IFN-γ). HCV-specific CD8⁺ T cellscan be detected by, for example, ⁵¹Cr release assays (see Example 2).⁵¹Cr release assays measure the ability of HCV-specific CD8⁺ T cells tolyse target cells displaying an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, orNS3NS4NS5aNS5b epitope. HCV-specific CD8⁺ T cells which express IFN-γcan also be detected by immunological methods, preferably byintracellular staining for IFN-γ after in vitro stimulation with an NS3,an NS4, an NS5a, or an NS5b polypeptide (see Example 3).

[0103] HCV-specific CD4⁺ cells activated by the above-described fusions,such as an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, expressed invivo or in vitro, and combinations of the individual components of theseproteins, preferably recognize an epitope of an NS3, NS4, NS5a, or NS5bpolypeptide, including an epitope of an NS3NS4NS5a or NS3NS4NS5aNS5bfusion protein, that is bound to an MHC class II molecule on anHCV-infected cell and proliferate in response to stimulating NS3NS4NS5aor NS3NS4NS5aNS5b peptides.

[0104] HCV-specific CD4⁺ T cells can be detected by alymphoproliferation assay (see Example 4). Lymphoproliferation assaysmeasure the ability of HCV-specific CD4⁺ T cells to proliferate inresponse to an NS3, an NS4, an NS5a, or an NS5b epitope.

[0105] Methods of Activating HCV-Specific T Cells.

[0106] NS3NS4NS5a fusion proteins or polynucleotides and NS3NS4NS5aNS5bfusion proteins or polynucleotides, or combinations of the individualcomponents of these proteins and polynucleotides, can be used toactivate HCV-specific T cells either in vitro or in vivo. Activation ofHCV-specific T cells can be used, inter alia, to provide model systemsto optimize CTL responses to HCV and to provide prophylactic ortherapeutic treatment against HCV infection. For in vitro activation,proteins are preferably supplied to T cells via a plasmid or a viralvector, such as an adenovirus vector, as described above.

[0107] Polyclonal populations of T cells can be derived from the blood,and preferably from peripheral lymphoid organs, such as lymph nodes,spleen, or thymus, of mammals that have been infected with an HCV.Preferred mammals include mice, chimpanzees, baboons, and humans. TheHCV serves to expand the number of activated HCV-specific T cells in themammal. The HCV-specific T cells derived from the mammal can then berestimulated in vitro by adding HCV NS3NS4NS5a or NS3NS4NS5aNS5bepitopic peptides to the T cells. The HCV-specific T cells can then betested for, inter alia, proliferation, the production of IFN-γ, and theability to lyse target cells displaying NS3NS4NS5a or NS3NS4NS5aNS5bepitopes in vitro.

[0108] In a lymphoproliferation assay (see Example 4), HCV-activatedCD4⁺ T cells proliferate when cultured with an NS3, NS4, NS5a, NS5b,NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absenceof an epitopic peptide. Thus, particular NS3, NS4, NS5a, NS5b,NS3NS4NS5a and NS3NS4NS5aNS5b epitopes that are recognized byHCV-specific CD4⁺ T cells can be identified using a lymphoproliferationassay.

[0109] Similarly, detection of IFN-γ in HCV-specific CD8⁺ T cells afterin vitro stimulation with the above-described fusion proteins, orindividual components of these proteins, can be used to identify NS3,NS4, NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b epitopes thatparticularly effective at stimulating CD8⁺ T cells to produce IFN-γ (seeExample 3).

[0110] Further, ⁵¹Cr release assays are useful for determining the levelof CTL response to HCV. See Cooper et al. Immunity 10:439-449. Forexample, HCV-specific CD8⁺ T cells can be derived from the liver of anHCV infected mammal. These T cells can be tested in ⁵¹Cr release assaysagainst target cells displaying, e.g., NS3NS4NS5a NS3NS4NS5aNS5bepitopes. Several target cell populations expressing differentNS3NS4NS5a or NS3NS4NS5aNS5b epitopes can be constructed so that eachtarget cell population displays different epitopes of NS3NS4NS5a orNS3NS4NS5aNS5b. The HCV-specific CD8⁺ cells can be assayed against eachof these target cell populations. The results of the ⁵¹Cr release assayscan be used to determine which epitopes of NS3NS4NS5a or NS3NS4NS5aNS5bare responsible for the strongest CTL response to HCV. NS3NS4NS5a fusionproteins or NS3NS4NS5aNS5b fusion proteins which contain the epitopesresponsible for the strongest CTL response can then be constructed usingthe information derived from the ⁵¹Cr release assays.

[0111] An NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein or polynucleotideencoding such a fusion protein, as well as the individual components ofthese fusion proteins or polynucleotides, can be administered to amammal, such as a mouse, baboon, chimpanzee, or human, to activateHCV-specific T cells in vivo. Administration can be by any means knownin the art, including parenteral, intranasal, intramuscular orsubcutaneous injection, including injection using a biological ballisticgun (“gene gun”), as discussed above.

[0112] Preferably, injection of an NS3NS4NS5a or NS3NS4NS5aNS5bpolynucleotide, or a compositions containing a combination of theindividual components of the fusion polynucleotides, is used to activateT cells. In addition to the practical advantages of simplicity ofconstruction and modification, injection of NS3NS4NS5a or NS3NS4NS5aNS5bpolynucleotides results in the synthesis of an NS3NS4NS5a fusion proteinor NS3NS4NS5aNS5b, respectively, in the host. Similarly, administrationof the individual components of these polynucleotides, such as in acomposition consisting essentially of individual polynucleotidesencoding NS3, NS4, NS5a or a composition consisting essentially ofindividual polynucleotides encoding NS3, NS4, NS5a and NS5b, results inthe expression of the individual proteins in the host. Thus, theseimmunogens are presented to the host immune system with nativepost-translational modifications, structure, and conformation. Thepolynucleotides are preferably injected intramuscularly to a largemammal, such as a human, at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5or 10 mg/kg.

[0113] A composition of the invention comprising an NS3NS4NS5a fusionprotein or polynucleotide, an NS3NS4NS5aNS5b fusion protein orpolynucleotide, combinations of these fusions, or a combination of theindividual components thereof, is administered in a manner compatiblewith the particular composition used and in an amount which is effectiveto activate HCV-specific T cells as measured by, inter alia, a ⁵¹Crrelease assay, a lymphoproliferation assay, or by intracellular stainingfor IFN-γ. The proteins and/or polynucleotides can be administeredeither to a mammal which is not infected with an HCV or can beadministered to an HCV-infected mammal. The particular dosages of thepolynucleotides or fusion proteins in a composition or will depend onmany factors including, but not limited to the species, age, and generalcondition of the mammal to which the composition is administered, andthe mode of administration of the composition. An effective amount ofthe composition of the invention can be readily determined using onlyroutine experimentation. In vitro and in vivo models described above canbe employed to identify appropriate doses. The amount of NS3NS4NS5apolynucleotide used in the example described below provides generalguidance which can be used to optimize the activation of HCV-specific Tcells either in vivo or in vitro. Generally, 0.5, 0.75, 1.0, 1.5, 2.0,2.5, 5 or 10 mg of an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein orpolynucleotide, or of each of the individual components, will beadministered to a large mammal, such as a baboon, chimpanzee, or human.If desired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

[0114] Immune responses of the mammal generated by the delivery of acomposition of the invention, including activation of HCV-specific Tcells, can be enhanced by varying the dosage, route of administration,or boosting regimens. Compositions of the invention may be given in asingle dose schedule, or preferably in a multiple dose schedule in whicha primary course of vaccination includes 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand/or reenforce an immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose or doses after severalmonths.

[0115] III. Experimental

[0116] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Those of skill in the art will readily appreciate that theinvention may be practiced in a variety of ways given the teaching ofthis disclosure.

[0117] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

EXAMPLE 1

[0118] Production of NS3NS4NS5a Polynucleotides.

[0119] A polynucleotide encoding NS3NS4NS5a (approximately amino acids1027 to 2399, numbered relative to HCV-1) (also termed “NS345a” herein)or NS5a (approximately amino acids 1973 to 2399, numbered relative toHCV-1) was isolated from an HCV. Polynucleotides encoding a methionineresidue were ligated to the 5′ end of these polynucleotides and thepolynucleotides were cloned into plasmid, vaccinia virus, and adenovirusvectors.

[0120] Immunization Protocols. In one immunization protocol, mice wereimmunized with 50 μg of plasmid DNA encoding either NS5a or encoding anNS3NS4NS5a fusion protein by intramuscular injection into the tibialisanterior. A booster injection of 10⁷ pfu of vaccinia virus (VV)-NS5a(intraperitoneal) or 50 μg of plasmid control (intramuscular) wasprovided 6 weeks later.

[0121] In another immunization protocol, mice were injectedintramuscularly in the tibialis anterior with 10¹⁰ adenovirus particlesencoding an NS3NS4NS5a fusion protein. An intraperitoneal boosterinjection of 10⁷ pfu of VV-NS5a or an intramuscular booster injection of10¹⁰ adenovirus particles encoding NS3NS4NS5a was provided 6 weekslater.

EXAMPLE 2

[0122] Immunization with DNA encoding an NS3NS4NS5a fusion proteinactivates HCV-specific CD8⁺ T cells.

[0123]⁵¹Cr Release Assay. A ⁵¹Cr release assay was used to measure theability of HCV-specific T cells to lyse target cells displaying an NS5aepitope. Spleen cells were pooled from the immunized animals. Thesecells were restimulated in vitro for 6 days with the CTL epitopicpeptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO:1) from HCV-NS5a in thepresence of IL-2. The spleen cells were then assayed for cytotoxicactivity in a standard ⁵¹Cr release assay against peptide-sensitizedtarget cells (L929) expressing class I, but not class II MHC molecules,as described in Weiss (1980) J. Biol. Chem. 255:9912-9917. Ratios ofeffector (T cells) to target (B cells) of 60:1, 20:1, and 7:1 weretested. Percent specific lysis was calculated for each effector totarget ratio.

[0124] The results of the assays are shown in Tables 1 and 2. Table 1demonstrates that immunization with plasmid DNA encoding an NS3NS4NS5afusion protein activates CD8⁺ T cells which recognize and lyse targetcells displaying an NS5a epitope. Surprisingly the NS5a polypeptide ofthe NS3NS4NS5a fusion protein was able to activate T cells even thoughthe NS5a polypeptide was present in a fusion protein.

[0125] Similarly, Table 2 demonstrates that delivery of the NS3NS4NS5afusion protein to mice by means of an adenovirus vector also activatesCD8⁺ T cells which recognize and lyse target cells displaying an HCVNS5a epitope. Thus, immunization with either “naked” (plasmid) DNAencoding an NS3NS4NS5a fusion protein or adenovirus vector-encodedfusion protein can be used to activate HCV-specific T cells.

EXAMPLE 3

[0126] Immunization with DNA encoding an NS3NS4NS5a fusion proteinactivates HCV-specific CD8⁺ T cells which express IFN-γ.

[0127] Intracellular Staining for Interferon-gamma (IFN-γ).Intracellular staining for IFN-γ was used to identify the CD8⁺ T cellsthat secrete IFN-γ after in vitro stimulation with the NS5a epitopep214K9. Spleen cells of individual immunized mice were restimulated invitro either with p214K9 or with a non-specific peptide for 6-12 hoursin the presence of IL-2 and monensin. The cells were then stained forsurface CD8 and for intracellular IFN-γ and analyzed by flow cytometry.The percent of CD8⁺ T cells which were also positive for IFN-γ was thencalculated. The results of these assays are shown in Tables 1 and 2.Table 1 demonstrates that CD8⁺ T cells activated in response toimmunization with plasmid DNA encoding an NS3NS4NS5a fusion protein alsoexpress IFN-γ. Immunization with an NS3NS4NS5a fusion protein encoded inan adenovirus also results in CD8⁺ HCV-specific T cells which expressIFN-γ, although to a lesser extent than immunization with aplasmid-encoded NS3NS4NS5a fusion protein (Table 2). TABLE 1HCV-NS5a-Specific CD8+ T Cells in Mice Immunized with NS5a or NS345a DNA⁵¹Cr Release Assay Intracellular Staining for IFN-γ Percent SpecificLysis of Percent of CD8+ T Cells Targets* Positive for IFN-g** E:T NS5aDNA NS345a DNA NS5a DNA NS345a DNA ratio p214K9 — p214K9 — p214K9 —p214K9 — 60:1 77 5 66 6 20:1 61 4 49 2 1.74 0.26 1.18 0.40  7:1 29 1 294

[0128] TABLE 2 HCV-NS5a-Specific CD8+ T Cells Primed by Adenovirus orDNA Encoding for NS345a ⁵¹Cr Release Assay Intracellular Staining forIFN-γ Percent Specific Lysis of Percent of CD8+ T Cells Targets*Positive for IFN-g** E:T NS345a Adeno NS345a DNA NS345a Adeno NS345a DNAratio p214K9 — p214K9 — p214K9 p214J p214K9 p214J 60:1 76 2 55 5 20:1 852 22 3 3.24 0.13 0.25 0.09  7:1 62 <1 10 3

EXAMPLE 4

[0129] Immunization with DNA encoding an NS3NS4NS5a fusion proteinstimulates proliferation of HCV-specific CD4⁺ T cells.

[0130] Lymphoproliferation assay. Spleen cells from pooled immunizedmice were depleted of CD8⁺ T cells using magnetic beads and werecultured in triplicate with either p222D, an NS5a-epitopic peptide fromHCV-NS5a (2224-AELIEANLLWRQEMG-2238; SEQ ID NO:2), or in medium alone.After 72 hours, cells were pulsed with 1 μCi per well of ³H-thymidineand harvested 6-8 hours later. Incorporation of radioactivity wasmeasured after harvesting. The mean cpm was calculated.

[0131] As shown in Table 3, immunization with a plasmid-encodedNS3NS4NS5a fusion protein stimulates proliferation of CD4⁺ HCV-specificT cells. Immunization with an adenovirus vector encoding the fusionprotein also resulted in stimulated proliferation of CD4⁺ HCV-specific Tcells (Table 4). TABLE 3 HCV-NS5a-Specific CD4+ T Cells in MiceImmunized with NS5a or NS345a DNA Mean CPM NS5a DNA NS345a DNA p222Dmedia p222D media 4523 740 4562 861 (x6.1) (x5.3)

[0132] TABLE 4 HCV-NS5-Specific CD4+ T Cells Primed by Adenovirus or DNAEncoding for NS345a Mean CPM NS345a Adeno NS345a DNA p222D media p222Dmedia 896 357 1510 385 (x2.5) (x3.9)

EXAMPLE 5

[0133] Efficiency of NS345a-encoding DNA Vaccine Formulations to primeCTLs in mice.

[0134] Mice were immunized with either 10-100 μg of plasmid DNA encodingNS345a fusion protein as described in Example 1, with PLG-linked DNAencoding NS345a, described below, or with DNA encoding NS345a, deliveredvia electroporation (see, e.g., International Publication No. WO/0045823for this delivery technique). The immunizations were followed by abooster injection 6 weeks later of 1×10⁷ pfu vaccinia virus encodingNS5a, plasmid DNA encoding NS345a or plasmid DNA encoding NS5a each asdescribed in Example 1.

[0135] PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymerswere obtained from Boehringer Ingelheim, U.S.A. The PLG polymer used inthis study was RG505, which has a copolymer ratio of 50/50 and amolecular weight of 65 kDa (manufacturers data). Cationic microparticleswith adsorbed DNA were prepared using a modified solvent evaporationprocess, essentially as described in Singh et al., Proc. Natl. Acad.Sci. USA (2000) 97:811-816. Briefly, the microparticles were prepared byemulsifying 10 ml of a 5% w/v polymer solution in methylene chloridewith 1 ml of PBS at high speed using an IKA homogenizer. The primaryemulsion was then added to 50ml of distilled water containing cetyltrimethyl ammonium bromide (CTAB) (0.5% w/v). This resulted in theformation of a w/o/w emulsion which was stirred at 6000 rpm for 12 hoursat room temperature, allowing the methylene chloride to evaporate. Theresulting microparticles were washed twice in distilled water bycentrifugation at 10,000 g and freeze dried. Following preparation,washing and collection, DNA was adsorbed onto the microparticles byincubating 100 mg of cationic microparticles in a 1 mg/ml solution ofDNA at 4 C for 6 hours. The microparticles were then separated bycentrifugation, the pellet washed with TE buffer and the microparticleswere freeze dried.

[0136] CTL activity and IFN-γ expression were measured by ⁵¹Cr releaseassay or intracellular staining as described in examples 2 and 3respectively. The results are shown in Table 5.

[0137] Results demonstrate that immunization using plasmid DNA encodingfor NS345a to prime mice results in activation of CD8+ HCV specific Tcells. TABLE 5 Efficiency of NS345a-Encoding DNA Vaccine Formulations toPrime CTLs in Mice ICS for IFN-gamma (% CD8+ cells that are IFN-g+) foldincrease NS345a DNA # of mice % # of vs. ‘naked’ CTL Vaccines Boost MeanSdtdevP tested responding expts DNA activity? NS345a DNA VVNS5a 1.021.70 41 68% 10 N/A YES NS345a DNA NS345a DNA 0.02 0.04 22  5% 5 N/A YESNS345a DNA NS5a DNA 0.22 0.21 24 63% 5 N/A YES NS345a DNA VVNS5a 5.004.36 7 100%  2 4.90 YES eV (electro- poration) PLGNS345a VVNS5a 2.652.54 6 100%  2 2.60 YES DNA PLGNS345a NS5a DNA 0.33 0.24 15 80% 3 1.50YES DNA

EXAMPLE 6

[0138] Immunization routes and replicon particles SINCR (DC+) encodingfor NS345a

[0139] Alphavirus replicon particles, for example, SINCR (DC+) wereprepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were injected with 5×10⁶ IU SINCR (DC+) repliconparticles encoding for NS345a intramuscularly (IM) as described inExample 1, or subcutaneously (S/C) at the base of the tail (BoT) andfoot pad (FP), or with a combination of ⅔ of the DNA delivered via IMadministration and ⅓ via a BoT route. The immunizations were followed bya booster injection of vaccinia virus encoding NS5a as described inExample 1.

[0140] IFN-γ expression was measured by intracellular staining asdescribed in Example 3. The results are shown in Table 6. The resultsdemonstrate that immunization via SINCR (DC+) replicon particlesencoding for NS345a by a variety of routes results in CD8+ HCV specificT cells which express IFN-γ. TABLE 6 Immunization Routes and SINCR (DC+Replicon Particles Encoding NS345a (all mice VVNS5a challenged) ICS forIFN-gamma (% CD8+ cells that are IFN-g+) # of % Immuniza- mice # ofresponding Vaccines tion Route Mean SdtdevP tested expts mice SINCR 100%IM 1.11 0.63 3 1 100% (DC+) (ta) 5 × 10⁶ SINCR 100% S/C 0.62 0.29 3 1100% (DC+) (BoT + FP) 5 × 10⁶ SINCR 2/3 IM (ta) + 2.43 2.00 3 1 100%(DC+) 1/3 S/C 5 × 10⁶ (BoT)

EXAMPLE 7

[0141] SINCR (DC+) vs SINDC (LP) replicon particles encodingfor NS345a

[0142] Alphavirus replicon particles, for example, SINCR (DC+) and SINCR(LP) were prepared as described in Polo et al., Proc. Natl. Acad. Sci.USA (1999) 96:4598-4603. Mice were immunized with 1×10³ to 1×10⁷ IU ofSINCR (DC+) or SINCR (LP) replicon particles encoding for NS345a, byintramuscular injection into the tibialis anterior, followed by abooster injection of 10⁷ pfu vaccinia virus encoding NS5a at 6 weeks.

[0143] IFN-γ expression was measured by intracellular staining asdescribed in Example 3. Administration of an increase in the number ofSINCR (DC+) replicon particles encoding NS345a resulted in an increasein % of CD8+ T cells expressing IFN-γ.

EXAMPLE 8

[0144] Alphavirus replicon priming, followed by various boostingregimes.

[0145] Alphavirus replicon particles, for example, SINCR (DC+) wereprepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were primed with SINCR (DC+), 1.5×10⁶ IU repliconparticles encoding NS345a, by intramuscular injection into the tibialisanterior, followed by a booster of either 10-100 μg of plasmid DNAencoding for NS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶IUSINCR (DC+) replicon particles encoding NS345a, or 10⁷ pfu vacciniavirus encoding NS5a at 6 weeks.

[0146] IFN-γ expression was measured by intracellular staining asdescribed in Example 3. The results are shown in Table 7. The resultsdemonstrate that boosting with vaccinia virus encoding NS5a DNA resultsin the strongest generation of CD8+ HCV specific T cells which expressIFN-γ. Boosting with plasmid encoding NS5a DNA also results in a goodresponse, while lesser responses are noted with adenovirus NS345a orSINCR DC+boosted animals. TABLE 7 Alphavirus Replicon Particle Priming,Followed by Various Boosting Regimens ICS for IFN-gamma (% CD8+ cellsthat are IFN-g+) # of % mice # of responding Vaccines Boost Mean SdtdevPtested expts mice SINCR NS5a DNA 0.46 0.36 4 1 75% (DC+) 1.5 × 10⁶ SINCRAdeno 0.04 0.04 4 1 25% (DC+) NS345a 1.5 × 10⁶ (10 × 10¹⁰) SINCR SINCR0.06 0.06 8 2 25% (DC+) (DC+) 1.5 × 10⁶ 1.5 × 10⁶ SINCR VVNS5a 2.43 2.454 1 100%  (DC+) (1 × 10⁷) 1.5 × 10⁶

EXAMPLE 9

[0147] Alphaviruses expressing NS345a

[0148] Alphavirus replicon particles, for example, SINCR (DC+) and SINCR(LP) were prepared as described in Polo et al., Proc. Nat. Acad. Sci.USA (1999) 96:4598-4603. Mice wee immunized with 1×10² to 1×10⁶IU SINCRDC+) replicons encoding NS345a via a combination of delivery routes (⅔IM and ⅓ S/C) as well as by S/C alone, or with 1×10² to 1×10⁶ IU SINCR(LP) replicon particles encoding NS345a via a combination of deliveryroutes (⅔ IM and ⅓ S/C) as well as by S/C alone. The immunizations werefollowed by a booster injection of 10⁷ pfu vaccinia virus encoding NS5aat 6 weeks.

[0149] IFN-γ expression was measured by intracellular staining asdescribed in Example 3. The results are shown in FIG. 1. The resultsindicate activation of CD8+ HCV specific T cells.

EXAMPLE 10

[0150] Efficiency of NS5a encoding DNA vaccine formulations to primeCTLs in mice Mice were immunized with either 10-100 μg of plasmid DNAencoding NS5a as described in Example 1 or with PLG-linked DNA encodingNS5a as described in Example 5. The immunizations were followed by abooster injection at 6 weeks of either 10-100 μg of plasmid DNA encodingfor NS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶ IU SINCR(DC+) replicon particles encoding NS345a, or 10⁷ pfu vaccinia virusencoding NS5a.

[0151] CTL activity and IFN-γ expression were measured by the methodsdescribed in Examples 2 and 3.

[0152] The results are shown in Table 8. The results demonstrate thatpriming with plasmid DNA encoding for NS5a or PLG-linked DNA encodingNS5a results in activation of CD8+ HCV specific T cells. TABLE 8Efficiency of NS5a-Encoding DNA Vaccine Formulations to Prime CTLs inMice ICS for IFN-gamma (% CD8+ cells that are IFN-g+) fold increase # ofmice % # of vs. ‘naked’ CTL NS5a Vaccines Boost Mean SdtdevP testedresponding expts DNA activity? NS5a DNA VVNS5a 1.67 1.49 8 100%  3 N/AYES NS5a DNA NS5a DNA 0.17 0.09 12 83% 3 N/A YES PLGNS5a NS5a DNA 0.220.09 9 100%  2 1.29 YES DNA NS5a DNA Adeno NS345a 0.10 0.08 4 50% 1 N/ANO NS5a DNA SINCR NS345a 0.20 0.17 4 75% 1 N/A YES

EXAMPLE 11

[0153] Efficiency of NS345b-encoding DNA vaccine formulations to primeCTLs in mice

[0154] Mice were immunized with 10-100 μg of plasmid DNA encoding NS34bby intramuscular injection to the tibialis anterior or with PLG linkedDNA encoding NS5a as described in Example 5. The immunizations werefollowed by a booster injection of plasmid DNA encoding for NS5a asdescribed in Example 1.

[0155] CTL activity and IFN-γ expression were measured by the methodsdescribed in Examples 2 and 3.

[0156] The results are shown in Table 9. The results demonstrate thatpriming with plasmid DNA encoding NS345b or PLG-linked NS345b results inactivation of CD8+ HCV specific T cells. TABLE 9 Efficiency ofNS345b-Encoding DNA Vaccine Formulations to Prime CTLs in Mice ICS forIFN-gamma (% CD8+ cells that are IFN-g+) fold increase NS345a DNA # ofmice % # of vs. ‘naked’ CTL Vaccines Boost Mean SdtdevP testedresponding expts DNA activity? NS345 DNA NS5a DNA 0.18 0.16 15 60% 3 N/AYES PLGNS345 DNA NS5a DNA 0.30 0.33 14 57% 3 1.67 YES

EXAMPLE 12

[0157] Administration of DNA via separate plasmids

[0158] Mice were immunized with 100 μg plasmid DNA encoding for NS345aor with 100 μg PLG-linked DNA encoding NS345a. Additionally, separateDNA plasmids encoding NS5a, NS34a, and NS4ab (33.3 μg each) wereadministered concurrently to another group of mice. Finally, PLG-linkedDNA encoding NS5a, NS34a, and NS4ab (33.3 μg each) were administeredconcurrently to another group of mice. The immunizations were followedby a booster injection of 1×10⁷ pfu vaccinia virus encoding NS5a, 6weeks post first immunization.

[0159] IFN-γ expression was measured by the method described in Example3. The results are shown in FIG. 2. The results demonstrate aparticularly vigorous response in the activation of CD8+ HCV specific Tcells when the DNA is broken down into smaller sub units and linked toPLG.

[0160] Thus, HCV polypeptides, either alone or as fusions, to stimulatecell-mediated immune responses, are disclosed. Although preferredembodiments of the subject invention have been described in some detail,it is understood that obvious variations can be made without departingfrom the spirit and the scope of the invention as defined by theappended claims.

1 2 1 9 PRT Artificial Sequence Description of Artificial Sequence CTLepitopic peptide from HCV NS5a 1 His Glu Tyr Pro Val Gly Ser Gln Leu 1 52 15 PRT Artificial Sequence Description of Artificial Sequence CD4+epitopic peptide from HCV NS5a 2 Ala Glu Leu Ile Glu Ala Asn Leu Leu TrpArg Gln Glu Met Gly 1 5 10 15

We claim:
 1. A fusion protein consisting essentially of an NS3, an NS4,and an NS5a polypeptide of a hepatitis C virus (HCV).
 2. A fusionprotein consisting essentially of an NS3, an NS4, an NS5a, and NS5bpolypeptide of an HCV.
 3. A fusion protein according to either of claims1 or 2, wherein one of the HCV polypeptides is derived from a differentstrain of HCV than the other HCV polypeptides.
 4. The fusion protein ofclaim 3 wherein each of the HCV polypeptides is derived from a differentstrain of HCV.
 5. A composition comprising: (a) a fusion proteinaccording to either of claims 1 or 2; and (b) a pharmaceuticallyacceptable excipient.
 6. A composition comprising: (a) a fusion proteinaccording to claim 4; and (b) a pharmaceutically acceptable excipient.7. A composition consisting essentially of: (a) an isolated and purifiedNS3 polypeptide of a hepatitis C virus (HCV); (b) an isolated andpurified NS4 polypeptide of a HCV; (c) an isolated and purified NS5apolypeptide of a HCV; and (d) a pharmaceutically acceptable excipientand optionally an adjuvant.
 8. A composition consisting essentially of:(a) an isolated and purified NS3 polypeptide of a hepatitis C virus(HCV); (b) an isolated and purified NS4 polypeptide of a HCV; (c) anisolated and purified NS5a polypeptide of a HCV; (d) an isolated andpurified NS5b polypeptide of a HCV; and (e) a pharmaceuticallyacceptable excipient and optionally an adjuvant.
 9. An isolated andpurified polynucleotide which encodes a fusion protein according toeither of claims 1 or
 2. 10. An isolated and purified polynucleotidewhich encodes the fusion protein of claim
 4. 11. A compositioncomprising: (a) the isolated and purified polynucleotide of claim 9; and(b) a pharmaceutically acceptable excipient.
 12. The composition ofclaim 11 wherein the polynucleotide is DNA.
 13. The composition of claim12 wherein the polynucleotide is in a plasmid.
 14. A compositioncomprising: (a) an isolated and purified polynucleotide according toclaim 10; and (b) a pharmaceutically acceptable excipient.
 15. Thecomposition of claim 14 wherein the polynucleotide is DNA.
 16. Thecomposition of claim 15 wherein the polynucleotide is in a plasmid. 17.A composition consisting essentially of: (a) an isolated and purifiedpolynucleotide encoding an NS3 polypeptide of a hepatitis C virus (HCV);(b) an isolated and purified polynucleotide encoding an NS4 polypeptideof a HCV; (c) an isolated and purified polynucleotide encoding an NS5apolypeptide of a HCV; and (d) a pharmaceutically acceptable excipientand optionally an adjuvant.
 18. The composition of claim 17 wherein thepolynucleotide is DNA.
 19. The composition of claim 18 wherein thepolynucleotide is in a plasmid.
 20. A composition consisting essentiallyof: (a) an isolated and purified polynucleotide encoding an NS3polypeptide of a hepatitis C virus (HCV); (b) an isolated and purifiedpolynucleotide encoding an NS4 polypeptide of a HCV; (c) an isolated andpurified polynucleotide encoding an NS5a polypeptide of a HCV; (d) anisolated and purified polynucleotide encoding an NS5b polypeptide of aHCV; and (e) a pharmaceutically acceptable excipient and optionally anadjuvant.
 21. The composition of claim 20 wherein the polynucleotide isDNA.
 22. The composition of claim 21 wherein the polynucleotide is in aplasmid.
 23. A method of activating T cells which recognize an epitopeof an HCV polypeptide, comprising the step of: contacting T cells with afusion protein of either of claims 1 or 2, whereby a population ofactivated T cells recognizes an epitope of the NS3, NS4, NS5a, or NS5bpolypeptides.
 24. The method of claim 23 wherein the T cells areobtained from a mammal selected from the group consisting of a mouse, ababoon, a chimpanzee, and a human.
 25. The method of claim 24 whereinthe mammal is infected with an HCV.
 26. The method of claim 24 whereinthe mammal is not infected with an HCV.
 27. The method of claim 23wherein the population of T cells comprises CD4⁺ T cells.
 28. The methodof claim 23 wherein the population of T cells comprises CD8⁺ T cells.29. The method of claim 28 wherein the CD8⁺ T cells expressinterferon-γ.
 30. The method of claim 28 wherein the CD8⁺ T cellsspecifically recognize an epitope of an NS5a polypeptide.
 31. The methodof claim 30 wherein the epitope is selected from the group consisting ofthe epitopes shown in SEQ ID NO:1 and SEQ ID NO:2.
 32. The method ofclaim 23 wherein the T cells comprise CD8⁺ and CD4⁺ T cells.
 33. Themethod of claim 23 wherein the step of contacting further comprisescontacting the T cells with an adjuvant.
 34. The method of claim 23wherein the fusion protein is provided by a polynucleotide encoding thefusion protein.
 35. The method of claim 34 wherein the polynucleotide isDNA.
 36. The method of claim 34 wherein the polynucleotide is RNA. 37.The method of claim 23 wherein the T cells are in a mammal.
 38. Themethod of claim 37 wherein the mammal is selected from the groupconsisting of a mouse, a baboon, a chimpanzee, and a human.
 39. Themethod of claim 37 wherein the mammal is infected with an HCV.
 40. Themethod of claim 37 wherein the mammal is not infected with an HCV.
 41. Amethod of activating T cells which recognize an epitope of an HCVpolypeptide, comprising the step of: contacting T cells with acomposition according to claim 7, whereby a population of activated Tcells recognizes an epitope of the NS3, NS4, NS5a, or NS5b polypeptides.42. A method of activating T cells which recognize an epitope of an HCVpolypeptide, comprising the step of: contacting T cells with acomposition according to claim 8, whereby a population of activated Tcells recognizes an epitope of the NS3, NS4, NS5a, or NS5b polypeptides.43. A method of activating T cells which recognize an epitope of an HCVpolypeptide, comprising the step of: contacting T cells with acomposition according to claim 17, whereby a population of activated Tcells recognizes an epitope of the NS3, NS4, NS5a, or NS5b polypeptides.44. A method of activating T cells which recognize an epitope of an HCVpolypeptide, comprising the step of: contacting T cells with acomposition according to claim 20, whereby a population of activated Tcells recognizes an epitope of the NS3, NS4, NS5a, or NS5b polypeptides.